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Natural Product ResearchFormerly Natural Product Letters

ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20

Anti-proliferative effect of pomegranate peelextracts on bovine peripheral blood mononuclearcells (PBMCs)

Fabio Mastrogiovanni, Annalisa Romani, Luca Santi, Nicola Lacetera &Roberta Bernini

To cite this article: Fabio Mastrogiovanni, Annalisa Romani, Luca Santi, Nicola Lacetera& Roberta Bernini (2019): Anti-proliferative effect of pomegranate peel extracts onbovine peripheral blood mononuclear cells (PBMCs), Natural Product Research, DOI:10.1080/14786419.2019.1627350

To link to this article: https://doi.org/10.1080/14786419.2019.1627350

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SHORT COMMUNICATION

Anti-proliferative effect of pomegranate peel extracts onbovine peripheral blood mononuclear cells (PBMCs)

Fabio Mastrogiovannia , Annalisa Romanib , Luca Santia , Nicola Laceteraa

, and Roberta Berninia

aDepartment of Agriculture and Forest Sciences (DAFNE), University of Tuscia, Viterbo, Italy;bDepartment of Statistics, Computing, Applications “Giuseppe Parenti” DiSIA, Phytolab, University ofFlorence, Florence, Italy

ABSTRACTPomegranate peel extracts prepared in our laboratories from awaste of juice fruit processing were tested on bovine peripheralblood mononuclear cells to evaluate the effects on viability, oxi-dative stress and proliferation. The (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl tetrazolium bromide) assay pointed out that the extractswere not cytotoxic at the tested concentrations (0.1, 1.0, and10mg/mL). A moderate protective effect against Reactive OxygenSpecies production induced by hydrogen peroxide or lipopolysac-charide and a significant anti-proliferative activity against prolifer-ation induced by concanavalin A were observed on cell linestreated with the extracts at 10lg/mL. Based on these results,pomegranate peel extracts seem promising as feed supplementfor dairy cattle, in particular around calving, when the animals aresubjected to an increase of the metabolic activity, responsible foroxidative stress and diseases. However, in vivo studies are neededto investigate the stability of the extracts across the bovinegastrointestinal tract barrier.

ARTICLE HISTORYReceived 26 March 2019Accepted 31 May 2019

KEYWORDSPomegranate peel extracts;agro-industrial by-products;polyphenols; peripheralblood mononuclear cells;anti-proliferative effect;circular economy

CONTACT Prof. Nicola Lacetera nicgio@unitus.itSupplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1627350.

� 2019 Informa UK Limited, trading as Taylor & Francis Group

NATURAL PRODUCT RESEARCHhttps://doi.org/10.1080/14786419.2019.1627350

1. Introduction

Plants are a precious source of valuable compounds exhibiting biological and pharma-cological effects as antioxidant, antimicrobial and anti-inflammatory activities. Forthese relevant properties, plant extracts have been used in the past against numerousdisorders and diseases, and currently they are object of intense studies to validatetheir potential as therapeutic agents in the promotion of health and disease preven-tion (D’Eliseo et al. 2019; Frezza et al. 2019; Mastrogiovanni et al. 2019).

Polyphenols are secondary metabolites variously distributed in the plant kingdom“derived from the shikimate/phenylpropanoid and/or the polyketide pathway, featur-ing more than one phenolic unit and deprived of nitrogen-based functions” (Yoshidaet al. 2016). They exhibit many beneficial effects for human health benefits includingthe antioxidant, anti-inflammatory and anticancer activity (Barontini et al. 2014; Berniniet al. 2015, 2018) and can be extracted from plants, fruits, and vegetables. When theywere recovered from agro-industrial waste and by-products using green extractivetechnologies, a virtuous process of reuse and valorization process with economic andenvironmental benefits can be realized according to the “circular economy” strategy(Bianco et al. 2006; Romani et al. 2016; Bernini et al. 2017).

Pomegranate (Punica granatum L.) is a fruit crop brought in the Mediterranean areaby the Phoenicians over 4000 years ago, which over the time has had a strongincrease. Fruits, peel and seeds are considerable sources of polyphenols as hydroxycin-namic acids, hydroxybenzoic acids, flavonoids and hydrolyzable tannins (Singh et al.2018; Russo et al. 2019).

Despite the wide number of in vitro and in vivo studies of pomegranate extracts todemonstrate the beneficial effects on human health, little is reported in the veterinary

2 F. MASTROGIOVANNI ET AL.

field. Based on this lack of literature, we recently planned to investigate the role ofnatural extracts to prevent the onset of the oxidative stress on dairy cattle during thecalving period, when the animals are subject to an increase of the metabolic activity(Sordillo and Aitken 2009) and to endocrine variations, which can produce immuno-suppression and serious diseases (Lacetera 2012).

In this context, we recently published a study on the activity of pomegranate peelextracts (PPE), rich of punicalagins on the oxidative stress and inflammatory statusinduced on bovine mammary epithelial cells, BME-UV1 (Mastrogiovanni et al. 2018).The experimental results have showed that, at non-cytotoxic concentrations (0.1, 1.0,and 10lg/mL), PPE produced a reduction of the oxidative stress and at 10lg/mL ofthe inflammatory status on BME-UV1 (Mastrogiovanni et al. 2018).

Based on these preliminary data, as a part of our research focused on the evalu-ation of of PPE as food supplement for dairy cattle, in this study we investigated theviability, the protective effect against Reactive Oxygen Species (ROS) productioninduced by hydrogen peroxide (H2O2) or lipopolysaccharide (LPS) and the anti-prolif-erative effect on bovine peripheral blood mononuclear cells (PBMCs) induced by con-canavalin A (ConA). To the best of our knowledge, this is the first study reporting theeffects of PPE on bovine PBMCs.

2. Results and discussion

PEE were obtained by a green extraction procedure based on the use of water(Imperatori et al. 2018). As depicted in Figure S1, the HPLC analysis evidenced thatPPE resulted rich of punicalagin (a- and b-isomer: 31.5 and 57.2% w/w, respectively)together to minor components as a- and b-punicalin (3.10% w/w), granatin B (1.48%w/w), ellagic acid and derivatives (6.25% w/w), gallic acid (0.38% w/w).

Then, the cytotoxicity of PPE on PBMCs was evaluated using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay at the concentrationspreviously tested by us on BME-UV1 (0.1, 1.0, and 10 lg/mL). The experimentaldata graphed in Figure 1 showed that any concentration resulted cytotoxicon PBMCs.

Therefore, the effects of PPE at these concentrations were evaluated on PBMCs afterROS production induced by H2O2 or LPS. After 48 h of treatment, PPE at 10 lg/mLshowed a protective effect against ROS production induced by H2O2 (�8.6%) or LPS(�17.5%); conversely, at the lowest concentrations, PPE did not show significant differ-ences compared to the control (Figure 2A).

Finally, we investigated the effect of proliferation in PBMCs induced by ConAand treated with 0.1, 1.0, and 10lg/mL of PPE. The Figure 2B evidenced a signifi-cant decrease of proliferation; in particular, PPE at 10mg/mL induced a reductionof 26%. In accordance with the literature data, this activity be could be relatedto punicalagin found in PPE (88.8%); in fact, it is described that this hydrolizabletannin is able to downregulate the IL-2 expression and cell proliferation via inhib-ition of NFAT activation showing a potent immune-suppressive activity (Leeet al. 2008).

NATURAL PRODUCT RESEARCH 3

3. Conclusions

This study has evidenced for the first time that PPE rich of punicalagins, obtainedfrom a by-product of the juice industry, exhibited biological effects on bovine PBMCs,reducing ROS production induced by H2O2 or LPS and proliferation induced by ConA.The protective effect against ROS production was modest while the anti-proliferativeactivity induced by ConA was significant. These data, combined to those obtained inour previous study (Mastrogiovanni et al. 2018), make PPE a promising ingredient offeed supplement in dairy cattle to prevent and counteract various immune systemderived pathologies, in particular during the calving period. However, in vivo studiesneeded to investigate the bioavailability of PPE in relation to the oral route of admin-istration and the stability across the bovine gastrointestinal tract barrier.

Figure 2. (A) ROS production after 48 h of treatment with PPE (0.1, 1.0, 10lg/mL) and 3 h ofinduction with H2O2 and LPS. (B) Cell proliferation after induction by ConA and co-treatment withPPE (0.1, 1.0, and 10lg/mL). Data (mean± standard error) are expressed as relative percentresponse respect to control level. (A) Cells treated with H2O2 or LPS; (B) cells treated with ConAare represented by horizontal dashed line. (�p< 0.05; ��p< 0.01; ���p< 0.001).

Figure 1. Cells viability after 48 h of treatment with PPE (0.1, 1.0, and 10lg/mL). Data (mean-± standard error) are expressed as relative percent response respect to control level; cells non-treated with PPE are represented by horizontal dashed line. (�p< 0.05; ��p< 0.01).

4 F. MASTROGIOVANNI ET AL.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

The authors gratefully acknowledge MIUR (MInistry for education, University and Research) forfinancial support (Law 232/216, Department of Excellence). The authors are also grateful toSIVAM Company (Casalpusterlengo, LO) that financed part of the research.

ORCID

Fabio Mastrogiovanni http://orcid.org/0000-0003-4858-7501Annalisa Romani http://orcid.org/0000-0003-0789-2120Luca Santi http://orcid.org/0000-0003-4638-5084Nicola Lacetera http://orcid.org/0000-0003-2088-2744Roberta Bernini http://orcid.org/0000-0002-2548-3876

References

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Bernini R, Gilardini Montani MS, Merendino N, Romani A, Velotti F. 2015. Hydroxytyrosol-derivedcompounds: a basis for the creation of new pharmacological agents for cancer preventionand therapy. J Med Chem. 58(23):9089–9107.

Bernini R, Carastro I, Palmini G, Tanini A, Zonefrati R, Pinelli P, Brandi ML, Romani A. 2017.Lipophilization of hydroxytyrosol-enriched fractions from Olea europaea L. by-products andevaluation of the in vitro effects on a model of colorectal cancer cells. J Agric Food Chem.65(31):6506–6512.

Bernini R, Barontini M, Cis V, Carastro I, Tofani D, Chiodo RA, Lupattelli P, Incerpi S. 2018.Synthesis and evaluation of the antioxidant activity of lipophilic phenethyl trifluoroacetateesters by in vitro ABTS, DPPH and in cell-culture DCF assays. Molecules. 23(1):208.

Bianco A, Bonadies F, Romeo G, Scarpati ML, Soriero A, Uccella N. 2006. The recovery of biophe-nols from wastewaters of olive oil production. Nat Prod Res. 20(3):259–264.

D’Eliseo D, Pannucci E, Bernini R, Campo M, Romani A, Santi L, Velotti F. 2019. In vitro studieson anti-inflammatory activities of kiwifruit peel extract in human THP-1 monocytes. JEthnopharmacol. 233:41–46.

Frezza C, Venditti A, Serafini M, Bianco A. 2019. Phytochemistry, chemotaxonomy, ethnopharma-cology, and nutraceutics of Lamiaceae. Stud Nat Prod Chem. 62:125–178.

Imperatori F, Barlozzari G, Scardigli A, Romani A, Macr�ı G, Polinori N, Bernini R, Santi L. 2018.Leishmanicidal activity of green tea leaves and pomegranate peel extracts on L. infantum.Nat Prod Res. 1.

Lacetera N. 2012. Effect of environment on immune functions. Environmental physiology of live-stock. The Atrium, Southern Gate, Chichester (England): Wiley-Blackwell Publications, 165–179.

Lee SI, Kim BS, Kim KS, Lee S, Shin KS, Lim JS. 2008. Immune-suppressive activity of punicalaginvia inhibition of NFAT activation. Biochem Biophys Res Commun. 371(4):799–803.

Mastrogiovanni F, Bernini R, Basiric�o L, Bernabucci U, Campo M, Romani A, Santi L, Lacetera N.2018. Antioxidant and anti-inflammatory effects of pomegranate peel extracts on bovinemammary epithelial cells BME-UV1. Nat Prod Res. 1.

NATURAL PRODUCT RESEARCH 5

Mastrogiovanni F, Mukhopadhya A, Lacetera N, Ryan MT, Romani A, Bernini R, Sweeney T. 2019.Anti-inflammatory effects of pomegranate peel extracts on in vitro human intestinal Caco-2cells and ex vivo porcine colonic tissue explants. Nutrients. 11(3):548–562.

Romani A, Pinelli P, Ieri F, Bernini R. 2016. Sustainability, innovation and green chemistry in the pro-duction and valorization of phenolic extracts from Olea europaea L. Sustainability. 8(10):1002.

Russo M, Cacciola F, Arena K, Mangraviti D, de Gara L, Dugo P, Mondello L. 2019.Characterization of the polyphenolic fraction of pomegranate samples by comprehensivetwo-dimensional liquid chromatography coupled to mass spectrometry detection. Nat ProdRes. 1.

Singh B, Singh JP, Kaur A, Singh N. 2018. Phenolic compounds as beneficial phytochemicals inpomegranate (Punica granatum L.) peel: a review. Food Chem. 261:75–86.

Sordillo LM, Aitken SL. 2009. Impact of oxidative stress on the health and immune function ofdairy cattle. Vet Immunol Immunopathol. 128(1–3):104–109.

Yoshida K, Cheynier V, Quideau S. 2016. Recent advances in polyphenol research, 5, 1–350. NewJersey, USA: John Wiley & Sons, Ltd.

6 F. MASTROGIOVANNI ET AL.

Supplementary Material

Anti-proliferative effect of pomegranate peel extracts on bovine

peripheral blood mononuclear cells (PBMCs)

Fabio Mastrogiovanni,a Annalisa Romani,

b Luca Santi,

a Nicola Lacetera,

a*

and Roberta Berninia

a Department of Agriculture and Forest Sciences (DAFNE), University of Tuscia,

Viterbo, Italy; b

Department of Statistics, Computing, Applications “Giuseppe Parenti”

DiSIA, Phytolab, University of Florence, Florence, Italy

Contact

*Prof. Nicola Lacetera, Department of Agriculture and Forest Sciences, University of

Tuscia, Via San Camillo de Lellis, 01100 Viterbo, Italy. E-mail: nicgio@unitus.it

Phone: +39 0761357581

Abstract. Pomegranate peel extracts prepared in our laboratories from a waste of juice

fruit processing were tested on bovine peripheral blood mononuclear cells to evaluate

the effects on viability, oxidative stress and proliferation. The (3-[4,5-dimethylthiazol-

2-yl]-2,5 diphenyl tetrazolium bromide) assay pointed out that the extracts were not

cytotoxic at the tested concentrations (0.1, 1.0 and 10 µg/mL). A moderate protective

effect against Reactive Oxygen Species production induced by hydrogen peroxide or

lipopolysaccharide and a significant anti-proliferative activity against proliferation

induced by concanavalin A were observed on cell lines treated with the extracts at 10

μg/mL. Based on these results, pomegranate peel extracts seem promising as feed

supplement for dairy cattle, in particular around calving, when the animals are subjected

to an increase of the metabolic activity, responsible for oxidative stress and diseases.

However, in vivo studies are needed to investigate the stability of the extracts across the

bovine gastrointestinal tract barrier.

Keywords: pomegranate peel extracts; agro-industrial by-products; polyphenols;

peripheral blood mononuclear cells; anti-proliferative effect; circular economy.

Experimental

Chemicals

All reagents were purchased from Sigma-Aldrich (Milan, Italy).

Extract preparation and chemical characterization

Pomegranate fruits (“Wonderful” variety) were collected in Grosseto (Tuscany, Italy;

Latitude: 42°45'46"N; Longitude: 11°06'33"E) in September 2017. Standardized peel

extracts were prepared as briefly described (Imperatori et al., 2018; Mastrogiovanni et

al., 2018). Fresh pomegranate peels were frozen with liquid nitrogen and crumbled in a

mortar to obtain a powder, which was treated with ultrapure water (w/v=1/12) (MilliQ

system waters Co., Milford, MA, USA) at 100 °C for 1h. After filtration, water was

removed by lyophilization (LYOVAC GT 2) and the extracts (PPE) were stored in a

laboratory dryer in amber glass bottles until the use.

A sample of PPE was analyzed by High Pressure Liquid Cromatography

(HPLC/DAD/ESI-MS) and Nuclear Magnetic Resonance (NMR). The

HPLC/DAD/ESI-MS analyses were carried out using a HP-1100 liquid chromatograph

equipped with a DAD detector and a HP 1100 MSD API-electrospray (Agilent

Technologies), a Luna C18 column 250×4.60 mm, 5 μm (Phenomenex) according to

already reported in the literature (Romani et al., 2012). The NMR spectra were recorded

using a 400 MHz Nuclear Magnetic Resonance spectrometer Advance III (Bruker,

Germany). Analytical data were detailed in our previous papers (Imperatori et al., 2018;

Mastrogiovanni et al., 2018).

Blood collection and PBMCs separation

Eight mid pregnant Holstein heifers (between 3 and 6 months of pregnancy) were used

as blood donors. Blood was collected by a vacutainer system (anticoagulant: sodium

heparin 10 IU/mL) from jugular vein.

After collection, blood samples were transferred to the laboratory using a portable

refrigerator. PBMCs were isolated as previously described (Lacetera et al., 2002).

Briefly, PBS (Sigma, St. Louis, MO) was added to all samples. Then, they were layered

on Ficoll-Paque PLUS medium (APB, Milan, Italy) and centrifuged at 600 x g and 18°C

for 45 min. The mononuclear cell band was recovered through glass pipettes and

washed twice in PBS. The red blood cells still present were removed with sterile-

distilled water causing hypotonic shock. The viability (typically over 90%) was

determined with trypan blue exclusion method, through automated cell counter

(Countess™ - Invitrogen™). PBMCs were suspended at 1 x 106 cells/mL of

concentration and were cultured in a culture medium composed by RPMI 1640 medium

containing 25 mM HEPES + 10% heat-inactivated fetal bovine serum, 2 mM l-

glutamine, 100 U of penicillin, 100 μg of streptomycin, and 0.25 μg of amphotericin

B/mL.

Cytotoxicity evaluation

The cytotoxicity of PPE at 0.1, 1.0, and 10 µg/mL was evaluated by the MTT [3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, after 48 h. Cells were

plated at 2 x 105 cells/well of concentration into 96-well plates (Sarstedt AG & Co.)

contained PPE dilutions or the culture medium (control) and later were kept at 39°C

humidified 5% CO2 incubator. After 48 h, plates were centrifuged at 300 x g for 10 min

then treatments were removed and replaced with 100 μL of solution composed by the

medium and MTT to a final concentration of 0.5 mg/mL. Then, after 4 h, plates were

again centrifuged at 300 x g for 10 min; supernatants were discarded and replaced with

100 μL of dimethyl sulfoxide; plates were additionally incubated for 45 min and finally

the absorbance at 595 nm was recorded using a microplates reader (Tecan Sunrise™).

ROS evaluation

ROS production was induced in PBMCs treating cells with PPE and adding hydrogen

peroxide (H2O2) or lipopolysaccharide (LPS) as direct and indirect oxidant agent,

respectively.22 Experimentally, 2 x 105 cells/well were plated in 96-well black plates

(Lumox®, Sarstedt AG & Co.) useful to fluorescence reading containing non-cytotoxic

PPE concentrations (0.1, 1.0, 10 µg/mL) or the culture medium (control). After 48 h, in

each well H2O2 (50 µM of final concentration) or LPS (20 µM of final concentration)

were added, then microplates were repositioned in incubator at 39°C. Three hours later,

plates were centrifuged at 300 x g for 10 min, all supernatants were discarded and

replaced by 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) 20 µM in PBS and

repositioned in incubator. After 40 minutes, the absorbance at 485 nm (excitation) and

535 (emission) was measured using a microplate reader (Ultimode Detector DTX 880,

Beckman Coulter Inc.).

Proliferation evaluation

The effect of PPE on the PBMCs proliferation induced by concanavalin A (ConA) was

evaluated using a commercial ELISA kit (Cell Proliferation ELISA, BrdU - Roche)

based on the determination of 5-bromo-2-deoxyuridine (5-BrdU) incorporated in the

DNA of cells in proliferation status. Experimentally, 2 x 105 cells/well were plated in

96-well plates (Sarstedt AG & Co.) containing non-cytotoxic concentrations of PPE

(0.1, 1.0, 10 µg/mL) or the culture medium (control). After addition of ConA, plates

were kept in incubator for 48 h. Then, 5-BrdU was added in each well and plates were

incubated for 48 h at 39°C. Finally, following the manufacturer’s instructions plates

were read at 450 nm by microplate reader (Tecan Sunrise™) and data were recorded.

Statistical analysis

All biological assays described were carried out in triplicate and data were converted in

percentages compared to the control. All data were analyzed by ANOVA using

Statistica 10 Software package (Stat Soft, Inc., USA) and are reported as mean ±

standard error. The significant of the differences was assessed by the Fisher’s Least

Square Difference (LSD) test and significance was declared at p< 0.05.

References

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Santi L. 2018. Leishmanicidal activity of green tea leaves and pomegranate peel

extracts on L. infantum. Nat Prod Res. doi:10.1080/14786419.2018.1481841

Lacetera N, Bernabucci U, Ronchi B, Scalia D, Nardone A. 2002. Moderate summer

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Biometeorol. 46: 33-37. doi:10.1007/s00484-001-0115-x.

Mastrogiovanni F, Bernini R, Basiricò L, Bernabucci U, Campo M, Romani A, Santi L,

Lacetera N. 2018. Antioxidant and anti-inflammatory effects of pomegranate peel

extracts on bovine mammary epithelial cells BME-UV1. Nat Prod Res.

doi:10.1080/14786419.2018.1508149.

Romani A, Campo M, Pinelli P. 2012. HPLC/DAD/ESI-MS analyses and anti-radical

activity of hydrolyzable tannins from different vegetal species. Food Chem. 130:

214-221. doi:10.1016/j.foodchem.2011.07.009.

Figure S1. Polyphenolic composition of PPE (Imperatori et al., 2018; Mastrogiovanni

et al., 2018)